Nuclear Magnetic Resonance Spectroscopy

Ian C. P. Smith* and Domthea E. Blandford

Institute for Biodiagnostics, National Research Council of Canada, 435 Ellice Avenue, Winnipeg, Manitoba, Canada R3B 1Y6

Nuclear magnetic resonance (NMR) spectroscopy is a powerful, nondestructive technique capable of complete structural andconformational analysis of complex molecules, quantitative analysis of complex mixtures, and noninvasive measurement of reactionrates of chemical systems in the test tube and in intact livingorganisms, including humans. NMR was discovered simultaneously by two independent laboratories in 1946 (Tl, T2).Subsequently, high-resolution NMR was quickly developed byanalytical chemists as a powerful technique for the determinationof molecular structure. The fundamentals of NMR technologyare described in specialty texts to which interested readers arereferred (T3-T6). In the first section of this review, we willprovide a brief overview of the basics of NMR spectroscopyincluding theory and principles of NMR, instrumentation, technicallimitations, and spectral interpretation methods. At this point weshall quickly shift to the designation MRS, magnetic resonancespectroscopy, which has been adopted by clinical proponents toavoid any negative implications of nuclear.The development of MRS as a clinical analytical tool has beenspurred on by the widespread use of a related analytical technique,magnetic resonance imaging (MRI), in clinical medicine. The twoare related since they both utilize the same physical phenomenon,NMR; MRS emphasizes spectral or chemical information, whereasMFU emphasizes spatial information. The techniques and applications of MRI will not be addressed in this review. Interestedreaders are referred to recent texts and articles (77-23).MRS is unique in its capability to provide nondestructive invivo and in vitro chemical analyses. While significant advanceshave been made in in vivo MRS, this has recently been reviewedelsewhere (TI@. The present review for clinical chemistryhighlights some of the research that has been directed toward invitro chemical analysis, namely, physiological fluids, tissue specimens, and tissue extracts. This application of MRS in pharmacology and toxicology is already quite well established; the main areasof current development are metabolic disorders, organ transplantation, neurological disorders, and cancer. The review covers thetime period of 1990 to the present. Our previous review coveredthe literature up to that time ( T l l ) .THE BASICS OF NMR SPECTROSCOPYTheory and Principles. Spectroscopy is the measurementof the frequency dependence of absorption or emission of energyby a system. NMR refers to the absorption and release of radiofrequency (rf) energy by a nucleus in a magnetic field. Possessionof both charge and spin renders some nuclei magnetic and confersvarious properties on them which affect their behavior in anexternal magnetic field. One such property is a magnetic moment@). In an external magnetic field (Bo),the magnetic moment ofa spinning nucleus will precess, or describe a cone, around thedirection of the field. The precessional frequency of a particularnucleus is proportional to the strength of the magnetic field.To observe resonance, the nuclei must be irradiated withelectromagnetic (I$radiation, the frequency of which must matchthe precessional frequency of the nuclei. The rf energy is thenabsorbed by nuclei in the lower energy spin state, raising themto the higher energy spin state. In actual fact, upward and

downward transitions are stimulated equally, but upward ones are

more prevalent due to the greater occupancy of the lower energystate. This leads to a net absorption of rf energy. Since theenergy difference between the two states is proportional to themagnetic field strength, the stronger the field, the greater is thedifference between the two populations, and the stronger is theMRS signal.For any particular atomic nucleus, at a constant magnetic fieldin a vacuum, there is only a single resonant frequency. In bulkmatter, nuclei are surrounded by electronic clouds which exert asmall, but significant shielding effect. The degree to which amagnetic nucleus is shielded from the applied field by the electroncloud determines its precessional frequency; the more dense isthe electron cloud (increased shielding), the lower is the precessional frequency. Different molecular environments are characterized by a parameter called the chemical shift, which is theresonance frequency measured relative to that of a suitablereference compound. The chemical shift values (6) are typicallyof the order ofand are therefore commonly specified in partsper million @pm). These units are independent of magnetic fields,thereby allowing direct comparison of results from differentinstruments. In absolute frequency terms, the separation betweennuclei with different chemical shifts increases with increasingmagnetic field, yielding better dispersion of resonances at highfield.Electronic clouds also mediate interactionsbetween nuclei thatresult in a characteristic splitting pattern of the MRS signal. Theso-called spin-spin couplings (usually given the symbol 7)arevery useful in assigning MRS resonances, but they also causespectra to be complicated and crowded. Nevertheless, thecharacteristic pattern of chemical shifts and couplings usuallyenables the identification and quantitation of each of a number ofcompounds present in a mixture.Consider the H MR spectrum of glucose in water, as shownin ref T12. Two major anomeric species of glucose are present,the a- and P-pyranose forms. The resonances at 4.51 and 5.10ppm are due to the hydrogens at position 1 of the pyranose ring,B and a anomers, respectively. Proximity to the oxygen in thering gives these hydrogens very characteristic chemical shifts.The splitting of these resonances into two is due to Jcoupling tothe hydrogen species at position 2 of the ring. The magnitude ofthe coupling is indicative of the coniiguration at position 1, 3.8Hz for the a-anomer and 8.0 Hz for the ,%anomer. The otherpatterns in the spectrum are more complex to analyze due tooverlaps, but assignments of all resonances and couplings for bothanomers have been made (T12).In the MRS experiment, after the rf pulse is turned off,relaxation processes occur to restore the original equilibrium. Therelaxation process implies a loss of energy from the system ofnuclear spins. Two different processes, both of which areessentially exponential, are characterized by time constants: TI,longitudinal relaxation, or spin-lattice relaxation, which resultsin a transfer of energy to the surroundings; and 72, transverserelaxation or spin-spin relaxation, which is a redistribution ofenergy among spins. Together 71and TZcan provide informationconcerning molecular conformation and dynamics.Analytical Chemistry, Vol. 67,No. 12,June 15, 1995

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In the NMR experiment, data are collected by a pulse/data

acquisition/delay sequence. The sequence is repeated to yieldan averaged free induction decay (FID) signal of adequate signal/noise ratio, which is subsequently Fourier transformed to yield aspectrum. The positions of NMR signals are measured in Hertzfrom a standard reference signal and are expressed in ppm inorder to be independent of the spectrometers field strength. Theintensity of an NMR signal is proportional to the area of the signal,and this area is generally measured by electronic integration.Under appropriate conditions, peak areas are proportional to theconcentration of the particular compound observed. Thus, formixtures of compounds, direct quantitative analysis, withoutseparation, is possible.It is evident that MRS could be a very powerful analytical toolin the clinical laboratory and is of vast potential for clinicalchemical analyses. However, this has not yet happened, perhapsdue to the cost of the instrumentation, certain technical limitations,and difficulties in data interpretation. Each of these issues willbe discussed in the following sections.Instrumentation. A modem MR spectrometer consists ofthree components: (1) a magnet, capable of sustaining a strong,stable, and homogenous magnetic field; (2) a probe within themagnetic field made up of a sample cavity, transmitter coil, andreceiver coil; and (3) appropriate electronic circuitry, a computer,and peripheral devices to detect, amplify and display the NMRsignal.(a) The Magnet. Magnets are classified as either permanentmagnets or resistive superconducting electromagnets. Becausethe precessional frequency of a nucleus is directly proportionalto the magnetic field strength, it is advantageous to use thestrongest possible magnetic field to obtain the greatest separationbetween signals. Recent advances in technology have resultedin increased spectral dispersion and sensitivity, simpliied spectra,and reduced interference from strong solvent signals. Thestrongest magnets, those which are superconducting, are nowused in MR instruments to generate field strengths up to 17.63 T(750 MHz for IH); permanent magnets and electromagnetsgenerate field strengths up to 2.1 (60 MHz) and 2.35 T (100 MHz),respectively. The superconducting magnet, a solenoid magnet,is a coil of wire (typically a niobium-titanium alloy) which isimmersed in liquid helium at a temperature of 4 K (-269 C). Atthis temperature, the material is superconducting and can carrya high current with no electric loss or heat generation. Thisrequires substantial thermal insulation. The superconductingmagnets are the most expensive to run; the principal running costsare in liquid gases, particularly liquid helium.(b) The Probe. At the center of the magnetic field is theprobe, which contains the sample cavity and the radio frequencycoil arrangement for excitation and detection of the signal. Othercoils are present in the magnet to generate additional smallmagnetic fields. Shim coils generate fields of various shapes sothat the magnetic field is homogeneous or uniform over the entiresample. The homogeneity results in narrow MR lines and thushigher spectral resolution. Further narrowing of MR lines canbe achieved by spinning the sample tube, which results in anaveraging of field inhomogeneities. Magnetic field stability (Le.,control of the strength of the magnetic field) is achieved andmaintained through an electronic technique known as fieldfrequency locking. This is accomplished by the selection of asubstance with a strong MR signal separate from those of the510R

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sample (e.g., deuterium for lH NMR). This substance can be kept

physically apart from the sample (external lock) or, more commonly, can be dissolved within the sample (internal lock). Thefrequency of the lock signal is electronically monitored andcompared to that of the magnetic field, and the magnetic field isautomatically and continuously adjusted, so that proportionalitybetween field and frequency is maintained.In a typical experiment, chemical samples of volume 0.1-10mL are placed in a narrow test tube. The test tube is thenpneumatically inserted into the center of the magnet. While onlyone sample can be analyzed at a time, automatic sample changersare now available.Commercially available NMR spectrometers are not inexpensive: modem computer controlled, multinuclear ITinstrumentsrange from $200 000 to $1500 000.Technical Limitations. The power of MRS lies in the largenumber of analytes potentially amenable to identification andquantitication by this method. Compounds that contain the atomicnuclei hydrogen (H), phosphorus CP), carbon (13C),sodium f3Na), and fluorine (19F) are most often used for clinical MRSapplications. With the exception of 19F,these nuclei are essentialcomponents of organisms and are present in almost 100%naturalabundance (except W ) . Each nucleus has a different frequencyof absorption (the chemical shift) in the NMR spectrum. Withrespect to MRS analysis of biofluids, most studies are performedusing lH or 31P. The lH nucleus has several significant advantages. It has the highest sensitivity of any stable nucleus, 100%natural abundance, and is found in virtually all metabolites. Ingeneral, H MRS of physiological fluids is hindered by threeproblems: (1) relatively low analytical sensitivity; (2) the aqueousnature of physiological fluids; and (3) the fact that physiologicalfluids are complex mixtures, resulting in difficulty in the resolutionand assignment of the large number of proton resonances. Theselimitations have all been largely overcome.(a) Analytical Sensitivity. MRS has a relatively low detectionsensitivity when compared with current routine clinical methodssuch as immunoassays, gas chromatography/mass spectroscopy,or the newer molecular diagnostic techniques. However, highresolution MRS has undergone many hardware developmentswithin the last decade. New high-field magnet technology, radiofrequency electronics, and novel probe designs have resulted inincreased spectral sensitivity and dispersion. The increase indetection sensitivity over the years for ethylbenzene, the compound used to evaluate spectrometers, has been impressive.Within a period of less than 35 years, the spectroscopy frequencyhas increased from 6 to 750 MHz, resulting in an increase in thesignal/noise ratio from 6 to 12 000. Given that time-averaging ofdata is often used to improve signal to noise ratios, the gain ofmore than 1000 in detection sensitivity translates into a decreaseof more than 106 in the time required to obtain a spectrum. MRsystems operating at 17.6 T, 750 MHz for H NMR, have recentlybeen delivered to customers. The sensitivity now attainableapproaches approximately 1 nM for H NMR (213). Moreover,relatively simple concentration techniques such as lyophilizationor Folch or perchloric acid extraction can be used to increasethe sensitivity for higher molecular weight compounds or analytesin low concentration. Of greater significance is the fact that MRSpossesses the potential to detect simultaneously a wide range ofcompounds of biological interest, irrespective of structure orphysical or chemical properties, resulting in an MRS profile or

fingerprint. This is in contrast with the majority of analytical

techniques currently used, which are designed to be specific fora single analyte or a particular class of analytes. The spectralprofile provided by H MRS can be of great utility for clinical anddiagnostic purposes, because changes in the normalprofile canreadily be detected in abnormal samples without the requirementthat the compound to be analyzed be preselected. Moreover,changes in analyte patterns in various physiological fluids maybe linked to pathological processes. This feature of MRS, coupledwith the accompanying structural information provided, has thepotential to detect novel markers of disease or toxicity withoutfirst having to predict their likely outcome.(b) Aqueous Nature of Physiological Fluids. The presenceof water-derived protons in biological fluids was an impedimentto studying solutes in aqueous solutions. Water-derived protonsare present in most body fluids at a concentration of approximately110 M, some 105 times the concentration of the metabolite ofinterest. Suppression of the water signal, which would otherwisedominate the spectrum, is therefore necessary, and a variety ofmethods have been described. These methods include selectivesaturation of the water signal by either the application of acontinuous or gated secondary irradiation field at the waterresonance frequency, or multipulse solvent suppression; e.g., theDante pulse sequence (Tl4); spectral selection based on TIrelaxation time (Le., the water elimination Fourier transformmethod, WEFT) (Tl5);approaches based on the augmentationof the water TZrelaxation time by the addition of a water protonexchange reagent or a paramagnetic agent (WATR method) andattenuation of the water signal by CPMG (Carr-Purcell-Meiboom-Gill) spin-echo methods (Tl6-Tl9);or a combination ofthese methods (T20).Most of these solvent suppression techniques are now straightforward in modern MR spectrometers and may result in acceptablesolvent suppression. In addition, the majority of data on physiological fluids were produced after the advent of these methods.Thus, while not a recent advance, a discussion has been includedin this review to highlight the fact that the water resonance isnot necessarily a limitation at this time. However, it must be keptin mind that these methods may still be inadequate for very dilutesolutions, and thus lyophilization of the specimen followed byredissolution in 2Hz0 is a practical method of eliminating the waterproblem and increasing the concentration of the metabolites ofinterest. Data obtained in this fashion must be interpreted withcaution, since this process can result in the loss of volatile orunstable compounds.(c) Physiological Fluids as Complex Mixtures. Severaldifferent methods have been applied to the analysis of MR spectra.In simple cases, the chemical shift values and intensities areautomatically obtained using peak-picking routines in the frequency domain spectrum. However, physiological fluids containa multitude of compounds, and thus an MR spectrum containsbroad resonances arising from macromolecules such as proteinsand lipids which may overlap resonances from species of lowmolecular weight. With the recent availability of higher fieldinstrumentation, higher frequency measurements decrease peakoverlaps and give more dispersed resonances. Nevertheless,resolution and identification of the resonances can be problematic.While the enormous complexity of the spectrum is indicative ofthe amount of biochemical information, useful, clinically relevantinformation is only obtained after elimination or reduction of these

broad resonances. One approach to this is convolution of the FID

with a function that discriminates against broad resonances suchas a sine bell convolution function. This function minimizes theinfluence of the broad underlying resonances due to macromolecules and enhances those due to species of low molecular weight.Thus, use of sophisticated methods facilitates spectral assignment.In the past, a variety of multidimensional techniques wereavailable, the most useful of which for clinical chemistry is twodimensional proton-proton shift correlation (2D-COSY) spectroscopy ( T l l ). In addition, solid-phase extraction chromatographywith off-line NMR detection provides a simple and efficient meansof separating and detecting complex mixtures of drugs andendogenous molecules (TZl).There are drawbacks to both ofthese techniques; the relatively long spectral accumulation timesrequired by the former, and the destruction of the biofluid matrixof the latter. Use of spin-echo pulse sequences or physicalpretreatment of the sample (T22)may be necessary to eliminatethe broad resonances arising from macromolecules. Recently,two-dimensionalJ-resolved ORES) spectroscopy has been shownto be an efficient means of extracting data on low molecular weightcompounds in urine and blood plasma (723,T23). Moreover,the plethora of biochemical information that is given by MRspectroscopy has led to the use of sophisticated pattern recognition methods for data compression and biochemical classification

(2-24-T28).CLINICAL APPLICATIONS OF NMRSPECTROSCOPYThe analysis of physiological fluids by high-resolution MRS isa relatively recent application of the NMR phenomenon in clinicalmedicine. Despite the fact that MR spectra are rich in informationon endogenous biochemical processes in health and disease, andthat quantitation of metabolites can be readily achieved, thediffusion of MRS methods into the clinical laboratory remains slow.While the major technical limitations have been overcome, MRSof physiological fluids competes with long-established biochemicalmethods that are well accepted, highly automated, comparativelyinexpensive, and readily available at most clinical sites. Moreover,data collection and interpretationis limited by the scarcity of MRStrained individuals able to exploit fully the wealth of informationin the spectrum of a fluid, tissue, or tissue extract.A large number of physiological fluids is accessible for MRSstudies in vitro. The first medical applications showing the utilityof H MRS analysis of complex metabolite mixtures involved theanalysis of urine and serum (224,T29,T30).A variety of otherfluids including cerebrospinal fluid (CSF), amniotic fluid, synovialfluid, sweat, aqueous humor, seminal plasma, saliva, bile, ascitesfluid, and tissue extracts have since been examined. All physiological fluids are not equally available in terms of quantity andease of availability (technical difticulties, patient benefit, patientdiscomfort, ethical issues). Nevertheless, samples are drawn ina variety of clinical situations where it would seem prudent toextract as much information as possible out of as small a specimenas possible, particularly in situations where a specimen is difficultto obtain. It is here where MRS can offer distinct advantages overconventional biochemical analytical techniques: MRS analysisrequires a small sample volume (0.2-0.5 mL), which generallyremains intact during measurement and thus can be used forsubsequent assay by other techniques; the specimen generallyrequires no or very little pretreatment; spectra take only a fewAnalytical Chemistry, Vol. 67,No. 72,June 75, 7995 511R

minutes to acquire and, unlike some other screening techniques

(e.g., HPLC), it is not necessary to preselect the metabolites ofinterest in order to detect and quantitate them.Presently, MRS has penetrated the areas of pharmacology andtoxicology. Many pharmaceutical companies have implementedautomated MRS screening procedures to aid the metabolic andtoxicological studies of various experimental therapeutic agents.MRS has also found applications in various clinically importantsituations. The main areas of current development are inhereditary metabolic disorders, organ transplantation, neurologicaldisorders, and cancer. These and other clinical applications willbe discussed in the following sections. The discussions willattempt not only to highlight the recent advances but also toemphasize the advantages of MRS over conventional analysis inparticular clinical situations.Hereditary Metabolic Disorders. Inborn Errors of Metabolism. Historically, one of the most successful clinical a pplications of MRS has been the detection of a wide range of inbornerrors of metabolism. In these disorders, the reduction or absenceof activity of a single enzyme or cofactor can have dramaticconsequences for metabolism and its control. Many inheritedmetabolic disorders result in the accumulation of large amountsof organic intermediates, or derivatives thereof, which are produced proximal to the defective enzyme step and eventually spillinto the blood and urine. 'H MRS has been used to study theurinary excretion of such compounds. The literature describing'H, 31P,and I3C studies was extensively reviewed by us (T22) andothers (T32). Most data on physiological fluids have been acquired on unmodified urine using 'H MRS. In the ensuing years,very little additional work has focused on this area. This is perhaps not surprising since this is essentially an application alreadysuited to routine use. Research energies have been directedtoward other clinical applications. However, recent investigationson unmodified urine have detected the metabolites histidine andformic acid in a patient with histidinemia (T3.2). The spectra wereacquired in 15 min and did not require any pretreatment of thespecimen. Similar investigations of urea cycle enzyme disordersdemonstrated the presence of the diagnostic metabolites citrullineand N-acetylcitrulline in four patients with citrullinemia (2'33);argininosuccinate in three patients with argininosuccinic aciduria(deficiency of argininosuccinate lyase (T33, T34)); and orotate,uracil, and uridine in four patients with ornithine carbamoyltransferase deficiency (7'33).Other studies have essentially confirmed the detection of the diagnostic metabolites in alkaptonuria(T32),multiple acyl CoA dehydrogenase deficiency (glutaric aciduria type 11) (T35),methylmalonic aciduria (T36),and propionicaciduria (T36). Recently it was shown that deproteinizing plasmasamples by centrifuging through a filter with a l@kDa molecularexclusion leads to improved quantification of metabolites, inparticular those associated with 5oxoprolinuria (T37).Neonatal screening programs currently exist for a number ofinborn errors of metabolism, in particular those for whichtreatment has been shown to prevent or ameliorate the severityof the disorder. Current test procedures routinely used includesimple chemical tests to detect excessive metabolites and aminoacids and various chromatographic techniques that range inpurpose from the detection of abnormal amino acids to thequantitative identification of specific amino acids. For eachparticular disorder, a different screening test is required, followedby confirmation using yet another diagnostic test. The develop512R

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ment of a screening program using a technology such as MRS

would allow one to analyze the same blood and/or urine samplesimultaneously for many markers. In fact, simultaneous measurement of such a range of components is not possible for othertechniques, and this coupled with the other benefits of MRSsuggests that MRS is a viable alternative to current neonatalscreening programs. Moreover, there exists the possibility forthe detection of some novel markers of inborn errors of metabolism, as well as insights into the underlying defects, using MRS.Organ Transplantation. High-field 'H MRS has recently beenused for the rapid multicomponent analysis of urine and plasmain order to investigate the patterns of metabolic changes associatedwith early rejection of transplanted kidneys (T38, T39) and hearts(T40-T4.2).(a) Kidney. The assessment of renal graft dysfunctionfollowing transplantation relies on the measurement of plasmacreatinine, renal biopsy, and response to therapy. Since novelmarkers of nephron damage, i.e., abnormal profiles of trimethylamine N-oxide (TWO), dimethylamine @MA) and dimethylglycine @MG), have been detected by MRS in urine and blood(T.2.2, T43),it seems reasonable to speculate that these or othermarkers may be able to detect early rejection processes requiringdialysis and/or cyclosporin toxicity or overdose. In a recent study(T38),the spectra of normal human urine showed signals forcreatinine, glycine, citrate, alanine, lactate, and N-methylatedmetabolites in the chemical shift range of 3.1-3.3 ppm. Thespectra of patients' urine collected following renal transplantationwere considerably different. Compared to normal urine, thespectral pattern of urine from a patient with an immediatefunctioning graft showed decreased concentrations of citrate andthe presence of high levels of protein. Urinary tract infection inanother patient was associated with an abnormal elevation ofalanine, glycine, lactate, acetate, and succinate. In a third patient,with renal tubular ischemia, elevated levels of the medullaryosmolytes, DMA and myo-inositol, and glucose were observed.The spectra of urine from a patient with a nonfunctioning graft,compared to that of normal urine, was grossly distorted as a resultof significant proteinuria and hematuria. Thus, profiles such asthese may provide diagnostic and prognostic information. Moreover, these spectral profiles suggest that the excretion of specificrenal metabolites may be associated with episodes of graftdysfunction. In this regard, a combination of parameters (e.g.,TMAO, DMA, alanine, citrate, etc.) all related to creatinineconcentration were recently studied. A high excretion of TMAOappeared to be associated with biopsy-confirmed acute graftrejection episodes (T38, T39). However, there was some degreeof overlap, suggesting that TMAO alone may not be a reliablemarker of graft dysfunction; further studies are required. Nevertheless, the results are encouraging and suggest that a combination of parameters in the proton spectra of urine could be used toimprove diagnosis and management of these patients. Thismethod could be routinely included in the evaluation of thesepatients with minimal inconvenience to the patient.(b) Heart Rejection of a heart transplant is similarly monitored by a combination of invasive and noninvasive techniquesincluding repeated endomyocardial biopsy and doppler echocardiography (CDE). Recently, two parameters measured by MRShave been proposed for use in the assessment of heart transplantrejection, plasma lipoproteins and the glycosylated residuesN-acetylglucosamine (NAG) and N-acetylneuraminic acid ("A)

borne by the plasma proteins. Changes in both these parameters

have previously been shown to reflect inflammatory processes andimmunological reactions. Measurement of the width at half-heightof both the methyl and methylene resonances arising fromlipoproteins in plasma (total line width, TLW) has been shown tobe correlated to graft rejection (T41);increased TLW values wereobserved in patients with evidence of a rejection process. Whena TLW cutoff value was set at 62 Hz, the sensitivity and specifcityof the test was 71 and 90%,respectively, with a positive predictivevalue (PPV) of 78%and a negative predictive value ( N w ) of 86%.If the TLW value was referred to a reference value (to minimizethe effects of a wide dispersion of preoperative TLW values), andthe ratio thereof was greater than 1.15,the accuracy of the TLWtest for detection of graft rejection increased. Sensitivity andspecifcity increased to 80 and 95%,respectively, with a P W of90%and a N W of 91%.The second parameter that has been proposed as a noninvasivemarker of rejection is the variation of the glycosylated residuesof glycoproteins NAG and NANA In a recent experiment, protonNMR spectra of plasma were obtained. Resonances were assignedto total glycosylated residues (GRt) and to mobile NAG and NANAresidues. Then GRt, NAG, and NANA were measured on thebasis of area of MRS signals (T40). The variations of the GRt/CH3 and (NAG NANA)/alanine ratios were analyzed singly, incombination with each other, and in combination with CDE, andcompared to an endomyocardial biopsy. Sensitivity was greatest(68%)when either the GRt/CH3 or (NAG NANA)/alanine ratioswere increased or CDE was positive; specifcity however was 51%.Specificity was greatest (95%) when the GRt/CHS or (NAGNANA)/alanine ratios were increased and CDE was positive;however, with this combination, sensitivity was decreased toapproximately 20%. Thus, the optimal detection requires acombination of the MRS and CDE parameters.Together, these preliminary studies are very encouraging andsuggest that analysis by proton MRS of blood plasma of hearttransplant recipients might contribute significantly to the earlydiagnosis of acute cardiac graft rejection. The advantages of aless invasive detection method are obvious, not only from thepatients point of view but also from a technical one. Biopsy, thegold standard, may not be accurate, since the pattern of rejectionwithin the myocardium is not necessarily uniform and may beasymmetric. Thus, the biopsy specimen may not be representativeof the underlying pathology. The ultimate objective may be tolimit the use of myocardial biopsy to confirmation of a previouslydetected rejection process.Neurological Disorders. The study of cerebrospinal fluidis a common aid to the differential diagnosis of neurologicaldiseases. CSF reflects the cytological and biochemical basis ofdisorders of the central nervous system and analysis thereofincreases the accuracy of the diagnosis. MRS allows the simultaneous quantitation of several metabolites that are not routinelymeasured in CSF and that would require several differentanalytical techniques to be assayed by conventional methods. Bythe combination of two-dimensional measurements, resonanceshave been assigned recently to 46 chemical species in CSF (T44).In addition, pattern recognition approaches and discriminantanalyses that separate samples into different classes have beenused in order to differentiate between normal controls and variousneurological disorders (T45-T47). It has been reported that thespectra of CSF of normal controls and subjects with tumors or

multiple sclerosis can be perfectly separated, whereas those from

subjects with disk herniations can be separated approximately 90%of the time using principle component analysis (T46). In thisstudy, spectra from various neurological disorders were compared.The spectrum of CSF from a normal subject showed signals forlactate, glucose, acetate, citrate and creatinine, the most importantmetabolites. In the spectrum of a case with an intramedullarymixed germ cell tumor, distinct differences were observed;glucose signals (3.2-4.0 ppm) were reduced, and new signalswere apparent between 0.8 and 1.0 ppm and at 1.45, 1.97, and2.39 ppm, identified as valine, a-alanine, and possibly putrescineand glutamine, respectively. Specimens from patients with diskherniations and multiple sclerosis differed from controls in therelative concentrations of acetate and a number of metabolites(including citrate, valine, a-alanine, acetate, creatinine, andglucose), respectively.Recent studies have addressed the issue of whether MRS ofCSF can be used as an aid in the diagnosis of specific neurologicaldisorders. Using high-resolution H MRS of human post mortemCSF, and pattern recognition computer methods, partial separationof CSF from patients with Alzheimers disease (AD) and normalcontrols was achieved (T48); more formal statistical analysissuggested that citrate by itself was the best discriminator. Citratelevels were signiscantly reduced in the CSF of AD patients relativeto control samples. These preliminary results are encouraging,since there are currently no antemortem diagnostic markers ofAD. Novel markers of AD may aid in the early diagnosis of AD,allowing affected patients the benefits of earlier therapeuticintervention.In the CSF of patients with Huntingtons disease (HD), MRSanalysis demonstrated a 60% increase in the pyruvic acid concentration as compared to controls; however, no unknown orunexpected metabolites were detected (T49).The significanceof this increase in pyruvic acid is unknown.A preliminary study of CSF from 30 patients with definite orsuspected multiple sclerosis (MS) showed that there were nosignificant differences between the levels of most metabolites ascompared to controls, with the exception of acetate and formate,which were increased and decreased, respectively, in patients withMS. Moreover, in 93%of patients with actively progressing MS,an unknown singlet peak at 2.82 ppm was found; this peak didnot appear in the spectrum of CSF from any of the control subjects(T50). The chemical shift suggested that the unknown is likelyan N-methyl metabolite, which may form the basis for a newdiagnostic test for MS. Such a test would be beneficial sincediagnosis of MS currently depends largely on its clinical features.Laboratory tests including increased IgG levels, oligoclonalbanding patterns on electrophoresis of CSF, increased levels ofmyelin basic protein, abnormal evoked potentials and lesions onMRI, and C T scans are useful only in support of the clinicaldiagnosis. A definitive marker would clearly be beneficial.The results obtained are promising and suggest that H MRSspectroscopy of CSF may result in an analytical tool for diagnosis,treatment monitoring, and prognosis of neurological disorders.Further prospective studies are warranted.Prenatal Diagnosis. MRS of human amniotic fluid is a recentclinical application of the NMR technology. Analysis of amnioticfluid can provide information on fetal and fetomatemal physiology.Indeed, amniotic fluid lipid measurements are used to identifyfetal lung maturity, amniotic fluid bilirubin measurements are usedAnalytical Chemistry, Vol. 67, No. 12, June 15, 1995

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to monitor the severity of erythroblastosis fetalis, and measurements of amniotic fluid a-fetoprotein and acetylcholinesterase areuseful in the prediction of open neural tube defects. Conventionalbiochemical tests have various technical limitations, and thus MRSmay be particularly well suited to the analysis of amniotic fluid.While high-resolution 'H MRS was first used to characterizeamniotic fluid for a variety of components including amino acids,lactate, and glucose (T52),recent work has generally focused ontwo areas, namely, 31PMRS analysis of phospholipid extracts ofamniotic fluid (T52-T54) and quantitation of the constituents ofamniotic fluid and their clinical correlation by 'H MRS (T55T57).(a) Fetal Lung Maturity. Testing for fetal lung maturity hastraditionally been done by the measurement of the lecithin/sphingomyelin ( W S ) ratio via numerous thin-layer chromatographic techniques. Novel techniques, such as fluorescencepolarization and lamellar body number counts, have been proposed recently to decrease technical difficulties and increaseturnaround time. In a preliminary study, 600-MHz 'H MR spectraof untreated amniotic fluid specimens from 66 patients wereanalyzed. Linear discriminant analysis was performed to determine how well the peak ratios could predict the fetal maturationcategory, as determined by either L/S ratio or fluorescencepolarization. Of 43 third-trimester fluids, 65%were placed in thecorrect category-immature, transitional, or mature (T55). Whilethis is a reasonable prediction of fetal lung maturity, it lacks thesensitivity and specificity required for a clinical test. However,the metabolites measured likely do not relate specifically topulmonary surfactant; better agreement might be anticipated whenmore relevant compounds such as phosphatidylcholine andphosphatidylglycerol are analyzed. 31P MR spectra of phospholipids in human amniotic fluid have been obtained recently (T52T54), but clinical correlations were not attempted.(b) Fetomaternal Complications. MRS of human amnioticfluid yields a wealth of information on chemical content and itsvariation with the condition of the mother (T58). To determinehow concentrations of the various metabolites of amniotic fluiddetected by MRS may relate to the clinical status of the fetus and/or the mother, a number of fetomaternal complications werestudied. No differences in peak intensity ratios were observedfor mothers with gestational diabetes or in cases of fetal trisomy21 where the spectra were generally normal in appearance (T55).Amniotic fluid from mothers with preeclampsia, on the other hand,showed differences in peak intensity ratios for choline, succinateand acetate. Linear discriminant analysis correctly distinguishedall cases (n = 5) of open spina bifida where the MRS spectra weremarkedly altered: lactate, glutamate, and acetate concentrationswere significantly increased. New peaks, previously not detectedin normal amniotic fluid were found, and other peaks normallypresent were absent (T55). Resonances observed at 6-8 ppm inthe MR spectrum of amniotic fluid have also been observed inthe same region in MR spectra of human urine. It has beensuggested that these resonances might be useful as markers forfetal renal output (T56). In addition, many low molecular weightcompounds in amniotic fluid have been reported to be of clinicalimportance: amino acid elevations have been reported in centralnervous system disorders (T59); glucose concentrations havebeen used in the diagnosis of intraamniotic infection (2'60);lacticacid has been associated with fetal acidosis (7'59). Thus, theability of MRS to provide a high-resolution spectrum with the514R

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identification of many low molecular weight constituents makes

this a powerful technique for the investigation of fetal lungmaturity and also conditions of suspected fetal anomalies and invarious maternal disease states. Moreover, in vitro MRS studiesof amniotic fluid may lay the foundation for noninvasive MRSanalysis of amniotic fluid in vivo.Infertility. Recently 'H MR spectroscopic methods have beenapplied to the analysis of seminal fluid and its componentsecretions from normal and infertile human males. The spectrumof whole seminal fluid is extremely complex with many overlapping resonances, but over 120 resonances have been assigned tovarious metabolites using a combination of 2-D 'H MRS methods(7'61) and a one-dimensional homonuclear polarization-transferexperiment (T62).In order to investigate whether the measurement of somebiochemical markers by MRS could allow the differentiation ofvarious types of infertility, Hamamah et al. (7'63) measured peakareas of glycerylphosphorylcholine (GPC) , glycerylphosphorylethanolamine (GPE), citrate, and lactate in seminal plasma ofnormal and infertile males. The peak areas for GPC, citrate, andlactate in seminal plasma were smaller for all infertile subjects ascompared to controls. In addition, peak area ratios for citrate/lactate, GPC/lactate, and GPE/GPC were found to be differentbetween infertile subjects with spermatogenic failure (nonobstructive) or obstructive azoospermia post vasectomy. With a cutoffvalue of 0.12, the GPE/GPC ratio was determined to have a highsensitivity (86%)and reasonable specificity (71%)in distinguishingbetween these two types of infertility. Other investigations haveshown that metabolite patterns differ between obstructive andnonobstructive azoospermia (T61). These results provide somequantitative markers that may have clinical applications in theevaluation of infertility in men using MRS, but more detailedstudies are needed.Cancer. Due to the increased incidence of cancer in recentyears, the search for an easy, accurate, and noninvasive screeningtest for early malignancy has inspired many investigations. In1986, a proton MRS measurement on human plasma that was quiteunlike the traditional tumor markers, such as a-fetoprotein orcarcinoembryonic antigen, was reported (T64). The averagemethyl and methylene resonance line width of the plasma 'H NMRspectrum, below 33 Hz, showed a strong correlation with thepresence of cancer. This initiated a large number of similarinvestigations which generally found this method to be unreliable(T65- T72), since it essentially measured hyperlipidemia. Asecond promising possibility for cancer screening has been thedetection of a novel lipoprotein band in density gradients of plasmafrom cancer patients (T73, T74). This band has been identifiedas lipoprotein(a) (T75).While these methods may not be suitablefor screening for early malignancy, they may yet develop a rolefor use as a tumor marker in the prognosis, monitoring oftreatment, and followup of patients previously diagnosed as havinga malignancy, particularly if serial measurements are made andconditions under which measurements are made are standardized.Alternatively, they may be regarded as complementary methodsin screening patients at risk for cancer, followup, and monitoringof treatment (T67, T76, T77).In recent years, attention has shifted away from the searchfor a simple screening test of plasma for malignant conditions tothe assessment of whether MRS of various tissue specimens andextracts of tissue specimens can differentiate between malignant

and nonmalignant diseases. The major focus in the literature has

been in cancer of breast, cervix, colon, liver, ovary, prostate, andthyroid, although investigations have looked at intracranial tumors(278, T79), endometrial carcinoma (T80),esophageal cancer(T81),lung cancer (T82),malignant melanoma (T83), andstomach cancer (T84).(a) Breast. MR spectroscopy of extracts from human breasttumors has utilized a number of nuclei. I3C NMR spectroscopydetected differences in the levels of monounsaturated and saturated fatty acids between carcinoma and noncancerous tissues(T85).The precise role of fatty acids in promoting breastcarcinoma and tumor progression has not been delineated, but apotential use of this technique includes screening for changes infatty acid composition that may predispose to development ofcarcinoma. The most numerous studies utilized 31PNMR spectroscopy to assess the role of phospholipid metabolites of tissueextracts. The results presently are inconclusive; some studiessuggest that phospholipid metabolite levels are not useful indicators of tumor prognosis (T86), whereas others suggest thecontrary (2'87, T88). Finally, proton MR spectroscopy demonstrated increased lactate content and an increased phosphocholinehaline ratio in tumor extracts, with decreased glucose andinositol, as compared to extracts of uninvolved tissue (T89).Quantitative MRS, or pattern recognition methods (vide infra),may provide even better means of correlating these findings withtumor characteristics such as grade and proliferative rate.(b) Colon. Detailed 'H MRS investigations of cancerous tissuebegan with colon. Early studies concentrated on the long TZvalueassociated with a lipid resonance at 1.3 ppm as an indicator ofmetastatic potential (290). Later studies demonstrated the utilityof particular peaks in the one- and two-dimensional 'H MR spectrato characterize stages of colon carcinoma (291). Recent studiesconfirm the utility of the method and suggest clinical screeningof colorectal biopsies could provide a useful adjunct to histologyfor the assessment of tissue intermediate between normal andmalignant (292). Detailed studies on colorectal cell lines ofvarying degrees of invasiveness support the earlier conclusionsand indicate correlations between genetic changes and theappearance of MR resonances (293).(c) Thyroid. Early studies of thyroid lesions suggested a rolefor lH NMR spectroscopic analysis of thyroid tissue in characterizing normal, benign, and malignant processes from one another(294). A recent study has supported such a role, demonstratingthat 'H MRS could separate thyroid neoplasms into two discrete(benign vs malignant) categories: benign follicular neoplasms,which are difficult to distinguish from their malignant counterpartsby histology, produced spectra with some parameters similar tothose of normal thyroid tissue, whereas malignant neoplasmsproduced spectra with properties in common with those ofpapillary, medullary, and follicular carcinomas (795). It hasrecently been shown, by means of consensus multivariate analysisof lH MR spectra, that benign follicular adenomas may bedistinguished from carcinomas with high sensitivity and spedicity,suggesting that many surgical interventions on thyroid may beeliminated by clinical use of 'H MRS (796).(d) Cervix. 'H MR spectra of cervical biopsies allow distinction between carcinoma of the cervix and cervical dysplasia (297).Using a convenient method of specimen preparation for lH MRsemiquantitative analysis, specimens could be grouped intonormal, dysplastic, and invasive cancer via simple MRS parameters

(298). Multivariate analysis of these spectra in our laboratory,

using methods reported in ref T96,indicates that subgroupingwithin the class of dysplasia is possible. It is hoped that thesemultivariate methods will be broadly used to extract the maximuminformation from the 'H MR spectra. Recently, using chemicalshift imaging based on the 1.3 ppm lipid resonance in the lH MRspectrum, it has been possible to map regions of carcinoma withina cervical biopsy (Ts9).(e) Ovary. Similar studies have recently been completed onovarian biopsies (TIOO). Malignant tissue could be distinguishedfrom normal and benign tissue with sensitivity and specificity of87 and 91%,respectively. Two-dimensional COSY spectra of thespecimens yielded classification with sensitivity and specificity of88 and 97%. In the two-dimensional spectra, cross-peaksindicativeof cell surface fucosylation were diagnostic for malignancy.(9Liver. Extracts of diseased liver tissues, including primaryand secondary tumors, and histologically normal tissue have beenstudied recently using both 31P and 'H MRS. The 31P MRspectrum of primary liver tumors showed an increase in phosphorylethanolamine and phosphorylcholine resonances and adecrease in glycerophosphorylethanolamineand glycerophosphorylcholiie when compared to spectra from histologically normaltissue (T101,T102).The 'H MR spectra of liver tissue showadditional and complementary information. Levels of severalmetabolites have been shown to change significantly in tumortissue compared with normal biopsies; citrate, alanine, lactate,taurine, and glycine are elevated,whereas creatine and threonineare decreased (T101).(g) Prostate. 31P,'H, and I3C MRS have been used in studiesto differentiate between benign and malignant lesions of theprostate gland. It has been suggested that the relative levels ofthe phosphorylated metabolites phosphocreatine and phosphomonoesters can be used to discriminate malignant from benign tissue(T103).Other investigators were able to measure the citrateconcentration in prostatic tissue using proton MRS (T104-TI06).Lower levels of citrate were not uniformly observed in cases ofadenocarcinoma as compared to benign prostatic hypertrophy,in particular mixed or primarily stromal hypertrophy (T105).However, relative ratios of metabolites (citrate/lactate, citrate/total choline, phosphocholine/total creatinine, choline/total creatine, alanine/total creatine, phosphoethanolamine/total phosphate, phosphocholine/total phosphate, and glycerophosphoethanolamine/total phosphate) were statistically different forprostate cancer specimens as compared to benign prostatichypertrophy (2'107).It is hoped that these observations maycontribute to the understanding of in vivo magnetic resonancespectra of the prostate and thus aid in the diagnosis of prostatemalignancy.Monitoring of Disease Processes or Therapy. Given thepotential of MRS for the diagnosis of a variety of pathologicalconditions, it follows that MRS would also have equal or greatervalue in the monitoring of either the disease processes or theresponse to a therapeutic regimen.(a) Inborn Errors of Metabolism. While not all inborn errorsof metabolism have effective treatment, dietary restrictions havebeen shown to ameliorate the symptoms of some of thesedisorders; for example, dietary restriction of the intake of phenylalanine and branched-chain amino acids have been advocated forthe treatment of phenylketonuria and branched chain ketoaciduria,respectively. These treatments require frequent and prolongedAnalytical Chemistry, Vol. 67, No. 12, June 15, 1995

515R

monitoring of the patients serum, which could be done by MRS

quickly and efficiently. Whether this has additional benefits overconventional methods is unclear at present; however, use of thistechnology may serve to provide novel markers of the disease ora greater understanding of the disease process. lH MRS has beenused to study metabolic perturbations in patients with disordersof propionyl-CoA metabolism (propionic acidemia and methylmalonic aciduria) during the administration of carnitine therapy.Administration of carnitine resulted in an increase in the excretionof propionylcarnitine and acetylcamitine coincident with animprovement in clinical condition (T108).(b) Premature and Sick Infants. The investigation of sickbabies is often complicated by the small sample volume that canbe collected for biochemical analysis. Often insufficient sampleis available to yield a complete picture of the biochemicalderangement. MRS has the advantage that the technique is rapidand nondestructive yet requires a small sample to give diversebiochemical information. Conditions of clinical relevance that havebeen previously monitored include drug metabolism, fasting,inherited metabolic disorders, birth asphyxia, necrotizing enterocolitis, and ketosis (T109).Recently, nutrient intake in prematureinfants was measured using MRS. An oral dose of D20 wasadministered, and urine samples were analyzed for DzO by HMRS. The results correlated well with the those of conventionalmethods, with the additional advantages of speed, accuracy, andease of sample preparation (Tll0). Thus, MRS investigations arepotentially useful in the diagnosis and monitoring of diseaseprocesses or response to treatment in sick or premature infants.(c) Transplanted Organs. As mentioned earlier, MRS hasbeen used to investigate early rejection processes and cyclosporintoxicity or overdose in patients with transplanted hearts orkidneys, and this area of investigation shows considerable promise(T38- T42).(d) Cancer Followup. While MRS of plasma and tissuespecimens has not given a specific tumor marker for diagnosis,recent work suggests that there may be suflicient differences inthe spectra for the effects of therapy to be monitored. This areais worthy of immediate investigation.(e) Renal Function. MRS studies on physiological fluids haveprovided much information on the biochemical and toxicologicaleffects of many compounds, as well as on endogenous biochemicalprocesses in health and disease. The application of MRS urinalysisto the study of the effects of region-specific nephrotoxins uncovered distinct abnormal patterns of metabolites that were associatedwith different sites of nephrotoxic action: renal proximal tubulartoxins caused glycosuria, lactic aciduria, and aminoaciduria; renalpapillary toxins produced different abnormal excretions patternsin terms of both time course and composition; and renal papillarynecrosis produced early increases in TMAO and DMA excretion,followed by subsequent increases in N,N-dimethylglycine, succinate, and acetate and decreases in TMAO and 2-oxoglutarate(Till). This knowledge of novel markers of site-specific renaldamage has been applied to the diagnosis of tubular and papillarydistortions in glomerulonephritis. Glomerulonephritis is characterized by an increase in the excretion of amino acid, ketonebodies, lactate, TMAO, and DMA and a decrease in the excretionof citrate and a-ketoglutarate (T212),indicating that tubularinterstitial changes and isolated tubular or papillary distortionsdevelop with the disease (T113).Moreover, these changes candevelop at any stage of the disease. Thus, MRS urinalysis is516R

Analytical Chemistry, Vol. 67, No. 72,June 75, 7995

suitable for diagnosing latent tubular interstitial changes, which

are not readily detected by traditional techniques and which resultin a poorer prognosis for the patient. This approach would enableidentification of patients at risk of rapid deterioration and wouldenable more aggressive treatment. Moreover, the effects of thetreatment could easily be monitored using this noninvasivemethod.Similar studies using plasma samples from patients withchronic renal failure demonstrated markedly elevated plasmacreatinine levels as well as an increase in lactate, TMAO, and DMA(TZZ,T43). The role of TMAO and DMA in the progression ofrenal failure has not been evaluated but may prove to be a markerof renal damage. Measurement of plasma creatinine, on the otherhand, is used clinically to assess glomerular filtration rate (GFR)and thereby evaluate the progression of renal disease or nephrotoxicity. Although plasma creatinine and creatinine clearancemeasurements are convenient to measure, they likely do notreflect GFR in patients with renal insufficiency. The clearance ofgandolinium (Gd) -diethylenetriaminepentaacetic acid (DTPA) ,an approved NMR contrast agent, has recently been evaluated asa novel marker of glomerular filtration. The results of theclearance of Gd-DTPA closely approximated the clearance oftechnetium [99MT~]DTPA, an accurate method for determiningGFR (2214). These results indicate that Gd-DTPA is a safe,nonradioactive indicator of GFR that may provide an alternativemethod for clinical studies of progressive renal disease.(f) Strength Recovery after Surgery. Watters et al. (2115)have followed the recovery of subjects from abdominal surgeryand correlated the degree of success with body composition andnitrogen balance. Pivotal to the study was the estimation of totalbody water by ingestion of D20 and analysis of its concentrationin urine by 2H MRS.SUMMARY AND CONCLUSIONSThe range of problems in clinical chemistry that can beaddressed by MRS is wide. The number of applications reportedin the literature is growing steadily, particularly since the studyof the composition of physiological fluids and tissues, and thechanges thereof in disease, are well suited to study by MRS.Moreover, the major technical limitations that have impeded itsprogress into the clinical laboratory in the past have beenaddressed. Recent hardware and software developments havefurther improved and simpliied MRS analysis. Thus, it wouldbe surprising if MRS of physiological fluids and tissues does notbecome an essential technique for clinical chemists and pathologists. In practice, three main obstacles remain to be overcome:a greater availability of instruments, a larger data base of spectralchanges correlated with pathological conditions, and an enhancedsupply of MR-trained individuals in the clinical environment.Ian C.P . Smith is Director General of the Institute for Biodiagnostics,National Research Council of Canada, in Winnipeg, Canada. He receivedB.Sc. (1961) and M.Sc. (1962) degrees in physacal chemistry from theUniversity ofManitoba and a Ph.D. (1965) an theoretical chemistryfiomthe University of Cambridge, England. A j e r postdoctoral research inbiophysics at Stanford University and the Bell Laboratories, he joined theDivision of Chemzstly, Natzonal Research Counczl %,Canada, Ottawa.He became the Director General of the Institute for aologacal Scaences,also in Ottawa, in 1987. His research interest is a plication of hysicaltechniques to the diagnosis, management, and un$rstanding ojhumandisease.Dorothea Blandford is a Scientific Associate of the Institute orBiodiagnostics, Nataonal Research Council of Canada, Winni eg. sfhereceived a B.Sc. de ree (1985) om the Unavers?ty of Waterio and aPh.D. degree (19 9 8 from the dave+y of Manatoba and Tom.leted apostdoctoral residency program an clznacal chemzstry (1993) an dnnipeg,

Manitoba. Her research interests include the a plication of both hysical

and chemical techniques to the diagnosis an! management ojhumandisease, as well as the pharmacokinetics and pharmacodynamics of itstreatment.